Short, single-stranded nucleic acids often serve as important factors for determining a disease or bioterrorism agent. The detection of these nucleic acids has broad applications, such as in pathogen identification and disease diagnosis. Many platforms have been developed for detecting nucleic acid molecules with specific sequences, including polymerase chain reaction (PCR), bio-barcode-based detection and electrochemistry. PCR-based methods, in particular, have been largely used to detect nucleic acids in ultralow abundance. However, this approach requires labor-intensive procedures and cumbersome instrumentation. These limitations have created significant challenges in healthcare medication in developing countries and other resource-limited sites. For example, one of reasons of the Ebola virus outbreak in Africa was the lack of diagnostic facilities in their local hospitals and clinics. Thus, effective detection and diagnosis, suitable for low-resource settings, is of particular importance.
In recent years, the development of visual detection methods based on gold nanoparticles (AuNPs), silver nanoparticles and graphene oxide has increased rapidly because of their simplicity and the visual readouts produced. Numerous methods based on AuNP aggregation have been developed to detect DNAs/RNAs, proteins and metal ions. To improve its sensitivity, enzymatic or non-enzymatic DNA circuits were further employed. An AuNP-based assay using magnetic microparticles (MMPs) was developed as a magnetophoretic assay with a significantly reduced detection time and a simplified equipment requirement. However, although AuNPs are widely used, their modification is time-consuming and requires delicate protocols to stabilize their mono-dispersion. For example, the mono-dispersed AuNPs are sensitive to the ionic strength of the solution. Alteration of the ionic strength may result in undesirable aggregation, creating additional uncertainty in optimizing the assay sensitivity and repeatability and making it incompatible with complex environments such as bio-fluids. On the other hand, the intrinsic colour of a biological sample can create significant interference for colorimetric assays. Consequently, delicate preparation or biomarker purification may be required, which restricts the practicality of the assay.
In addition, the current bottleneck of visual detection sensitivity usually falls at a nanomoles per liter level and is insufficient for detecting target molecules in low abundance. Various amplification methods have been developed, e.g. enzymatic amplification. However, enzymatic amplifications require specific conditions for enzymatic reactions, e.g., thermal cycles, and special storage for protein enzymes that may be difficult for resource-limited sites. Biobarcode amplification uses target molecules to connect nanoparticles that carry a vast number of barcode DNAs. The barcode DNAs, whose amount is proportional to the target molecules, are subsequently dissociated from the nanoparticles and detected, resulting in amplified signals representing the target molecules. However, the dissociation of biobarcode DNAs requires time-consuming dithiothreitol (DTT) treatment or toxic cyanides, which may increase complexity of the assays.
Accordingly, there remains a strong need for methods for determining the presence or amount of target nucleic acid sequences indicative of a health condition or a disease of an individual for subsequent diagnosis or prognosis based on a simple procedure, with low instrumentation requirements and sufficient sensitivity. In particular rapid and time-saving approaches are urgently required for specifically detecting the presence or amount of viral nucleic acid sequences and/or disease-indicative sequences in an individual so as to allow for an early diagnosis and early treatment of the individual. The present invention therefore provides novel approaches for such a purpose.